Identification of hydroxycinnamic acid–tartaric acid esters in wine by HPLC–tandem mass spectrometry

Food Chemistry 123 (2010) 827–833

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Identification of hydroxycinnamic acid–tartaric acid esters in wine by HPLC–tandem mass spectrometry F. Buiarelli *, F. Coccioli, M. Merolle, R. Jasionowska, A. Terracciano Dipartimento di Chimica, Università ‘‘Sapienza”, P.le Aldo Moro, 5-00185 Rome, Italy

a r t i c l e

i n f o

Article history: Received 23 March 2009 Received in revised form 22 February 2010 Accepted 3 May 2010

Keywords: Wine Hydroxycinnamic acid–tartaric acid esters Biophenolic compounds Polyphenols HPLC–MS/MS

a b s t r a c t In this work hydroxycinnamic acid esters of tartaric acid (mono-p-coumaroyl tartaric acid, monocaffeoyl tartaric acid, monoferuloyl tartaric acid esters) were separated, identified and quantified in several red wines by high performance liquid chromatography coupled with tandem mass spectrometry. The method features direct analysis of wines with no preparation and analysis of grape juice with minimum sample manipulation. For the identification was used both a triple quadrupole and a quadrupole–Tof, while for the quantification a triple quadrupole. At the same time two impurities, due to interfering substances coming from methanol (stored in bottles with polypropylene tops without Teflon protection), were characterised. These impurities had the same molecular weight as the investigated compounds. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Esters of hydroxycinnamic acids are widely distributed in plant world and in ‘‘vitis vinifera” grapes (Ong & Nagel, 1978). From a chemical point of view they belong to the class of biophenolic compounds that play an important role in wine quality. They contribute to sensory attributes such as bitterness, astringency, flavour and colour (Andrade et al., 2001). Moreover from the scientific community they are recognised as being responsible for several beneficial physiological effects on human health for their antioxidant properties (Monagas, Bartolomè, & Gomez-Cordoves, 2005; Saenz, Garcia, Ahumada, & Ruiz, 1989). The presence of these compounds is also important in enology in order to study origin and quality of wine (Gambelli & Santaroni, 2004). In grape ripening cinnamil tartaric acids, cinnamic acids and benzoic acids undergo variation of concentration, reaching a maximum value in August and then decreasing (Paronetto, 1977). During fermentation their content changes and the browning capacity of the grapes is modified (Ough & Amerine, 1988). In particular the effect of fermentation, storage, and fining on the content of hydroxycinnamoyl tartaric acids has been evaluated in Pinot blanc wines (Vrhovsek & Wendelin, 1998). In a previous work the identity of the hydroxycinnamic acid–tartaric acid esters were established in grape juice through hydrolysis of esters followed * Corresponding author. Tel.: +39 06 49913645; fax: +39 06 490631. E-mail address: [email protected] (F. Buiarelli). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.017

by gas–liquid chromatography (GC) equipped with flame ionisation detector or by high pressure liquid chromatography with UV detection (Ong & Nagel, 1978). In another study (Buiarelli et al., 1995), it was underlined that retention times (tr) of these compounds were lower than those of correspondent free cinnamic acids (caffeic, p-coumaric, ferulic), and UV spectra were similar to those of respective cinnamic acids. The identity of these esters was obtained after alkaline hydrolysis and HPLC–UV analysis of the free phenolic acids of the hydrolisate. In 2002 varietal differences among the polyphenol profile of seven table grape cultivars were studied by LC–DAD–MS–MS (Cantos, Espin, & Tomas-Barberan, 2002). Recently the isolation of some hydroxycinnamoyl tartaric acids from grape pomace has been carried out by high speed counter-current chromatography (Maier et al., 2006). The aim of our work was to identify these substances by HPLC– MS/MS in red wines, comparing results obtained with a triple quadrupole mass spectrometer and a quadrupole/time of flight (Tof). Such identification was difficult because the standard compounds are not still commercially available and because of the occasional presence of solvent impurities with the same molecular weight of two of cinnamil tartaric acids. By HPLC-Q/Tof we identified three cinnamil tartaric acid and their exact molecular weight together with the origin of the contaminating substances and their formulae. By HPLC coupled with triple quadrupole in Multiple reaction monitoring (MRM mode) after identification cinnamil tartaric acid esters were quantitatively determined in several wines.

828

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

2. Materials and methods 2.1. Chemicals Methanol (HPLC grade), ammonium formate and ammonia solutions 30% (RPE grade) were obtained from Carlo Erba (Milan, Italy). Formic acid, Chlorogenic acid hemihydrate (3-caffeoyl quinic acid) were obtained from Fluka (Sigma–Aldrich, Milan, Italy). Cichoric acid (dicaffeoyl tartaric acid) was purchased by Apin Chemicals Limited (UK). Ultra-pure water was produced with Pure Lab System (USF Elga, Ransbac-Baumbach, Germany). Syringe driven filter unit (Millex-HN, nylon 0.45 lm) Millipore Corporation Bedford (MA 01730, USA) was used. Cartridge Strata X were purchased by Phenomenex (USA).

Ferulic, p-coumaric and 3,4,5-trimetoxycinnamic (internal standard I.S.) acids were purchased by Fluka (Switzerland), caffeic acid from Sigma (Milan, Italy). 2.2. Samples Wines: Ten red wines, whose nine were purchased in retail store (Lambrusco, Sangiovese, Sangiovese basilicata, Barbera, Chianti, Syrah, Corvo Rosso, Primitivo del Tarantino, Castelli Romani) and one homemade, were directly analysed by HPLC–MS/MS after filtration. Pressed grapes: Samples (available on market) of pressed red grapes (3 mL) were clarified by natural settling of the sediment under cold-winter effect, (such phenomenon caused precipitation of bitartrate or cremotartarate). Such a solution, after filtration on a

Fig. 1. (A) Q/Tof MS spectrum of dicaffeoyl tartaric acid in negative ionisation mode. Condition as in the text in Section 2.3. (B) MS/MS Q/Tof in negative ionisation mode of the precursor ion m/z = 311 corresponding to monocaffeoyl tartaric acid originating from cichoric acid. Condition as in the text in Section 2.3.

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

filter of 0.45 lm, passed through a Strata  cartridge and after washing it with distilled water, the content of the cartridge was eluted with 2 mL methanol, then evaporated under nitrogen to a final volume of 100 lL. 2.3. Instrumentation LC–MS analysis was carried out by Shimadzu two pumps system LC-10 AD (Shimadzu, Kyoto, Japan) at a flow rate of 150 lL/min. The injector was Rheodyne 8125 system with a 5 lL sample loop. The

829

HPLC column (250 mm  2.1 mm I.D.) was slurry packed in our laboratory with Kromasil C18, 5 lm (EKA Nobel AB, S-44501 Surte, Sweden) (Benincasa et al., 1987). MS and MS/MS analyses were performed on a triple quadrupole PE-SCIEX API 365 (Perkin Elmer Sciex Instruments, Foster City, CA, USA), equipped with Turboion Spray interface in negative ion mode. The API source voltage was set between 3500 and 4500 V. The orifice potential (OR) was optimised between 3 and 80 and the ring protection (RNG) from 200 to 370 V. Collision gas (N2), curtain gas (N2) and nebulising gas (air) were set at 3 and 8, 8 (arbitrary units). The collisional

Fig. 2. Product ions of quasi-molecular ion m/z 311, 295, 325 obtained by MS/MS infusion with triple quadrupole in ion spray negative mode after collection and dilution of the pressed sample to 1:10, corresponding respectively to monocaffeoyl tartaric acid, mono-p-cumaroyl tartaric acid and to monoferuloyl tartaric acid.

830

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

energy was adjusted by variation of the voltage difference between the high pressure entrance quadrupole (Qo) and the collisional cell quadrupole (R02) and was found to give highest sensitivity for these analytes at 18 eV. The vaporiser was set at 350 °C. Data acquisitions were performed first, on each standard compound by direct continuous infusion (20 ng/lL) in full scan by adding ammonium formate 2 mM, in negative mode using the first quadrupole to chose an abundant precursor. MS/MS product ion scans were then recorded. Finally, all the analyses on standards and the real samples were carried out by HPLC–MS/MS in MRM (multiple reaction monitoring) mode (sample control) monitoring the product ions selected from MS/MS spectra to obtain a high specificity and sensitivity. The mobile phase was methanol + HCOOH 0.09% and H2O + HCOOH 0.09% in gradient elution at a flow of 150 lL/min. In parallel HPLC analysis was carried out also by Waters system 1525 (Waters, Milford, USA). The injection valve was Rheodyne model 8125 with a 5 lL sample loop. The column (250 mm  2.1 mm I.D.) was Kromasil. The elution was carried out in gradient as above.

MS and MS/MS analyses were performed on a quadrupole/Tof (Q/Tof) mass spectrometer (Micromass, Manchester, UK) equipped with a Z spray source interface in negative mode. Instrument’s operation, data acquisition and analysis were performed using Mass Linx 4.0 software (Micromass) on a windows server. The cone voltage, extraction voltage, microchannel plate (MCP) detector voltage and collision energy were optimised for each compound. Nitrogen was used as a nebulizer and curtain gas, at respectively 700 and 50 l/h when using HPLC. Desolvation temperature was 350 °C and source temperature 120 °C. Initially acquisition of MS parameters were optimised in ion spray mode by direct continuous infusion of a standard solution of the analytes (20 ng/lL in methanol–ammonium formate 2 mM) at a flow rate of 10 lL/min in the mass spectrometer by ESI-MS and then by ESI-MS/MS. The Micromass Q/Tof is a high resolution mass spectrometer that enables automatic exact mass measurements. The instrument also features a quadrupole mass filter and collision cell for MS/MS analyses. This powerful combination delivers simple exact mass measurements of fragment ions to

Table 1 Precursors and selected fragments of some cinnamic and cinnamil tartaric acids in negative ion spray and their retention time (tr) in HPLC in negative ion spray. Last column: window experiments used for HPLC–MS/MS acquisition of hydroxycinnamic acid esters of tartaric acid and phenolic acids. Compounds

tr (min)

MW

Precursor (m/z)

Fragments (m/z)

Window A: 0–28 min; B: 28–45 min

Monocaffeoyl tartaric acid Mono-p-cumaroyl tartaric acid Monoferuloyl tartaric acid Chlorogenic acid Caffeic acid Dicaffeoyl tartaric acid p-Coumaric acid Ferulic acid 3,4,5-T.M.cinnamic acid (IS)

18.0 23.7 25.6 27.1 30.1 35.0 35.1 36.1 44.7

312.0 296.0 326.0 354.3 180.0 474.0 164.0 194.0 238.0

311.0 295.0 325.0 353.0 179.0 473.2 163.0 193.0 236.9

179.0, 163.1, 193.2 191.0 135.0 311.1, 119.0 149.0 178.1,

A A A A A and B B B B B

149.0 149.0

293.1

133.1, 103.1

Fig. 3. Total Ion Chromatogram (TIC) in ion spray in negative MRM mode of cinnamic acids and cinnamic acid esters of red wine injecting (5 lL) directly to HPLC after filtration through a 0.45 lm filter. Letters A and B refer to acquisition periods, as shown in Table 1.

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

yield increased confidence in structural elucidation and data bank search results.

3. Results and discussion 3.1. Identification Mono-p-coumaroyl tartaric acid (coutaric acid), monocaffeoyl tartaric acid (caftaric acid), and monoferuloyl tartaric acid (fertaric acid) are depside of cinnamic acids (caffeic, p-coumaric, ferulic) with tartaric acid. Since no such standards are commercially available, we firstly studied the mass spectrometric behaviour of two other naturally occurring depsides: chlorogenic acid (3-caffeoyl quinic acid) which is a depside of caffeic acid and quinic acid, and a standard of cichoric acid (dicaffeoyl tartaric acid). Both of them were analysed by

831

infusion in ion spray negative mode by triple quadrupole mass spectrometry and by Q/Tof. We obtained the quasi-molecular ion [M H] and their characteristics fragments in MS/MS. To analyse cinnamil tartaric acids we started from the same electrical parameters (optimisation of lenses, potentials of spectrometer and so on). Fig. 1 shows above (A), for example, the MS Q/Tof spectrum of dicaffeoyl tartaric acid in negative ionisation mode. In the full scan spectrum the quasi-molecular ion [M H] m/z = 473 is evident, but also the fragments m/z = 311 and 179 due respectively to caffeoyl tartaric acid and caffeic acid moieties. With cichoric acid we managed to obtain a quasi-molecular ion of monocaffeoyl tartaric acid and its respective fragments. We assumed the other two cinnamil tartaric acids having the same behaviour. Fig. 1 shows below (B) MS/MS Q/Tof in negative ionisation mode choosing as precursor the ion m/z = 311 corresponding to monocaffeoyl tartaric acid originating from cichoric acid. In parallel the fragments were confirmed by triple quadrupole.

Fig. 4. MRM chromatogram of the acquisition period A (time 0–28 min) of Fig. 3, containing experiments and transitions described in Table 1.

832

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

taric acid esters, was first analysed with Q/Tof and in parallel with triple quadrupole. Fig. 2 shows fragments of quasi-molecular ions m/z 311, 295, 325 obtained by MS/MS infusion with triple quadrupole in ion spray in negative mode after dilution of the pressed sample to 1:10, corresponding respectively to: monocaffeoyl tartaric acid, mono-p-cumaroyl tartaric acid and monoferuloyl tartaric acid. Table 1 summarises precursors, main fragments and HPLC retention time for each investigated analyte and the experiments used in HPLC–MS/MS in MRM in sample control mode (Bianco et al., 2003). The table is divided into two time windows (A, B), on the basis of the acquisition period used in MS/MS analysis. Retention time of caffeic acid is about 30 min, so it is the first analyte acquired in window B. The acquisition of caffeic acid also in the first part of the chromatogram (window A) helped us to identify in a sample a peak at tr = 18 min. The origin of this peak in real sample is due to a more complex molecule containing caffeic acid with eventual sugars attached to the molecule. Fig. 3 shows the Total Ion Chromatogram (TIC) in ion spray in negative MRM mode of cinnamic acids and cinnamic acid esters of red wine injecting (5 lL) directly to HPLC after filtration through a 0.45 lm filter. Letters A and B refer to acquisition periods, as shown in Table 1. In particular, Fig. 4 shows the MRM chromatogram of the acquisition period A (time 0–28 min) of Fig. 3, containing experiments and transitions described in Table 1.

At the same time we purified by preparative HPLC–UV cinnamil tartaric acids from a wine sample. Collection of cinnamil tartaric ester peaks were made on the basis of their retention times and UV spectra, as described before (Buiarelli et al., 1995), as Rt are shorter compared to correspondent free cinnamic acids and UV spectra (carried out by photodiode detector) are very close to respective cinnamic acids. First of all the collected fractions were analysed by HPLC–MS in SIM mode (choosing m/z = 295 of monoferuloyl tartaric acid, m/ z = 311 of monocaffeoyl tartaric acid and m/z = 325 of mono-pcumaroyl tartaric acid), but no signal for these compounds was obtained at the predicted retention times. This was probably due to an excessive dilution of the sample and to a high limit of detection (LOD) of the technique. In contrast at the end of chromatogram (tr = 63.1) min we noticed the presence of several interfering signals with the same m/z ions (data not shown). For these analyses we used gradient elution (mobile phase methanol–distilled water added by formic acid of 0.09% to both solvents) by varying the composition of methanol from 0% to 100% in 25 min. Unfortunately after several experiments we realised that there was a contamination of methanol (Carlo Erba plus for HPLC) in contact with bottle-tops of polypropylene without Teflon protection, or due to use of glass-containers with propylene stoppers. The infusion by Q/Tof of ‘‘polluted” methanol, after exposition to sunlight, brings to three main peaks with different m/z ratio: 339.3, 325.3 and 311.2. Such substances were analysed by HPLC–Q–Tof and then the exact mass was calculated. The ions 311, 325, 339 correspond (with a minimum error of 5 ppm) to brute formulae C20H24O3, C21H28O3, C22H28O3. After Q/Tof MS/MS experiment product ion spectra were obtained starting from the above precursor (data not shown).

3.2. Quantitation results The amount of the cinnamil tartaric acid in wines is low and the pure standards of the investigated analyte are not always commercially available. So the concentration of the analytes was determined from purified solution of cichoric acid or considering the response of correspondent acid compound in methanol (with the same mass spectrometric behaviour). Five calibration levels (0.5, 5, 25, 50 and 80 ng/lL) in double aliquots, were used to build the calibration curves; 5 lL of extracts were injected at least three times each. The relative calibration graphs are given respectively

3.1.1. Pressed grapes To find a solution containing such substances in highest possible concentration we prepared, as described above, samples of pressed grapes. Such sample, after collection of the cinnamil tar-

Table 2 Quantitative results of each analyte in different red wines by HPLC–MS/MS (triple quadrupole). Wine

Compound (mg/L CV) Mono-p-cumaroyl tartaric acid

p-Coumaric acid

Monoferuoyl tartaric acid

Ferulic acid

Monocaffeoyl tartaric acid

Caffeic acid

Lambrusco

2.2 10

0.6 9.6

0.7 10.1

– –

4.0 9.3

3.1 7.6

Sangiovese

19.3 5.3

19.3 4.3

5.4 8.7

– –

6.0 7.9

70.7 3.3

Sangiovese basilicata

17.1 5.6

4.8 7.7

2.6 8.2

2.9 7.7

7.9 6.9

8.1 5.3

Barbera

23.1 6.3

1.5 9.3

3.6 7.5

1.1 9.2

53.8 2.6

6.7 9.5

Chianti

19.9 5.1

3.3 11.1

3.2 9.0

1.0 8.7

36.6 4.5

11.3 5.6

Homemade

20.5 6.8

11.9 6.8

6.5 7.1

2.0 10.0

59.4 3.2

6.9 5.5

Syrah

7.4 9.7

11.7 5.9

3.0 11.21

1.8 7.0

23.6 4.6

15.9 5.5

Corvo Rosso

8.8 7.5

3.2 8.2

4.4 5.8

1.4 11.1

15.5 4.7

22.5 4.0

Primitivo del Tarantino

27.1 4.8

4.1 7.6

5.8 7.3

1.6 7.0

77.4 2.5

20.3 5.0

Castelli Romani

10.5 6.2

5.8 7.6

2.2 6.7

1.2 10.2

28.9 5.4

6.7 7.2

Average (mg/L) MIN–MAX (mg/L)

15.6 2.3–27.1

6.6 0.6–19.3

3.7 0.7–6.5

1.3 0–2.9

31.9 4.0–77.4

17.2 3.1–70.7

F. Buiarelli et al. / Food Chemistry 123 (2010) 827–833

by the equations plotting against concentration the ratio of analyte area/ISTD area and the calibration line with R2 indicate a good linearity in the considered range concentration. Cichoric acid y = 0.0024x 0.0124 with R2 = 0.9992. Caffeic acid y = 0.0297x + 0.2343 with R2 = 0.9986. p-Coumaric acid y = 0.0535x + 0.1618 with R2 = 0.9979. Ferulic acid y = 0.0289x + 0.0619 with R2 = 0.9982. Monoferuloyl tartaric acid and mono-p-cumaroyl tartaric acid undergoes elution so close to each other that it is not possible to separate them perfectly, not even by preparative HPLC. The LOD was calculated as 3 times the ratio signal to noise and the LOQ as 6 times the LOD. The LOD of the acids were: caffeic acid, 0.3 mg/L; p-coumaric acid, 0.2 mg/L; ferulic acid, 0.3 mg/L. In establishing amounts of cinnamil tartaric acids the calibration line of the corresponding free acid was used. Table 2 shows the results obtained analysing nine commercial red wines and one homemade. It is reported the average value with the RSD % for the three hydroxycinnamic acid–tartaric acid esters and their respective free acids. The amount indicated in the table for the esters are an estimation because they differed by a constant from the true value, considering that the slope of the free acid lines are bigger than those of the correspondent cinnamil tartaric acids. Anyway this allowed us to monitor the trend of these substances in wines. At the end of the table it is summarised the average value of each analyte considering all the wines together and the minimum and maximum value. From this table it is evident that in most of the cases the ester is more abundant of the correspondent free acid and among cinnamil tartaric ester the most abundant was the monocaffeoyl tartaric acid followed by p-coumaroyl and then monoferuloyl tartaric acid. The same trend is followed by free cinnamic acids according to the data of other researchers (Kallithraka, Salacha, & Tzourou, 2009). 4. Conclusions This work is in line with the research about wines already developed in our laboratory. The cinnamil tartaric acids (monocaffeoyl tartaric, mono-p-cumaroyl tartaric and monoferuloyl tartaric acid) were identified in grapes and quantified in some red wines. Their determination, together with biophenols, is useful to monitor their variation during ripening of grapes, vinification and transformation that happens in wines with ageing. In order to cor-

833

relate these data it is necessary to determine all components or the highest possible number of every single class of non-volatile compounds by HPLC–MS/MS analyses, that are both rapid and reproducible in any laboratory equipped with such apparatus. The quantitative data of the esters were underestimated, but this allowed us to monitor the trend of these substances in wines. The predominant hydroxycinnamic acid was the caftaric acid followed by coutaric and then fertaric acid. In the identification of cinnamil tartaric esters we also detected, by MS/MS in negative ionisation mode, 3 characteristic impurities present in methanol coming from test-tube or from solvent bottle stoppers (with molecular weight 312, 326, 340) and these generated a fragment with m/z = 183.0. References Andrade, P. B., Oliveira, B. M., Seabra, R. M., Ferriera, M. A., Ferreres, F., & GarciaViguera, C. (2001). Analysis of phenolic compounds in spanish Albrarino and portuguese Alvarinho and Loureiro wines by capillary zone electrophoresis and high performance liquid chromatography. Electrophoresis, 22, 1568–1572. Benincasa, M., Cartoni, G. P., & Coccioli, F. (1987). Preparation and applications of microbore columns in HPLC. Annales de Chimie (Rome), 77(9–10), 801–811. Bianco, A., Buiarelli, F., Cartoni, G. P., Coccioli, F., Jasionowska, R., & Margherita, P. (2003). Analysis by liquid chromatography–tandem mass spectrometry of biophenolic compounds in olives and vegetation waters. Part I. Journal of Separation Science, 26, 409–416. Buiarelli, F., Cartoni, G. P., Coccioli, F., & Levetsovitou, Z. (1995). Determination of phenolic acids in wine by high performance liquid chromatography with microbore column. Journal of Cromatography A, 695, 229–235. Cantos, E., Espin, J. C., & Tomas-Barberan, F. A. (2002). Journal of Agricultural and Food Chemistry, 50(20), 5691–5696. Gambelli, L., & Santaroni, G. P. (2004). Polyphenols content in some Italian red wines of different geographical origins. Journal of Food Composition and Analysis, 17, 613–618. Kallithraka, S., Salacha, M. I., & Tzourou, I. (2009). Changes in phenolic composition and activity of white wine during bottle storage: Accelerated browning test versus bottle storage. Food Chemistry, 113, 500–505. Maier, T., Sanzenbacher, S., Kammerer, D. R., Berardini, N., Conrad, J., Beifuss, U., et al. (2006). Journal of Chromatography A, 1128, 61–67. Monagas, M., Bartolomè, B., & Gomez-Cordoves, C. (2005). European Food Research and Technology, 220, 331–340. Ong, B. Y., & Nagel, C. W. (1978). High-pressure liquid chromatographic analysis of hydroxy-cinnamic acid–tartaric acid esters and their glucose esters in Vitis vinifera. Journal of Chromatography, 157, 345–355. Ough, C. S., & Amerine, H. A. (1988). Methods for analysis of must and wine (2nd ed., pp. 168–169). New York: John Wiley and Sons. Paronetto, L. (1977). Polifenoli e Tecnica Enologica p. 84. Bologna: Edagricole. Saenz, H. T., Garcia, A. D., Ahumada, M. C., & Ruiz, M. (1989). Il Farmaco, 53, 448–449. Vrhovsek, U., & Wendelin, S. (1998). The effect of fermentation, storage, and fining on the content of hydroxycinnamoyltartaric acids and on browning of Pinot blanc wines. Wein-Wissenchaft, 53(2), 87–94.